Submitted to: DNA and Cell Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2005
Publication Date: 4/13/2006
Citation: Kwak, I., Song, S., Blum, J.L., Simmen, R.C., Simmen, F.A. 2006. Enhancer- and silencer-like sequences that mediate insulin-like growth factor-binding protein-2 gene expression in uterine cells of pregnancy. DNA and Cell Biology. 25(1):6-18.
Interpretive Summary: The ACNC is interested in how nutrition and dietary factors affect grow, development, and function of reproductive organs, including the mammary gland and uterus. We recently demonstrated that soy protein isolates positively influence the developing uterus, and further studies are necessary to determine how soy alters reproductive health of both the mother and the fetuses. In this study, we have identified unique characteristics of the pig IGFBP-2 gene that control its activity in the uterus during pregnancy. We found that this gene codes for the synthesis of a protein that causes uterine cells to divide during pregnancy to allow for this organ to grow so as to accommodate the growing fetus. Our results have identified how this is controlled in the nucleus and in so doing, may also explain why the IGFBP-2 gene is more active in cancer cells, where it promotes uncontrolled growth. Future studies can build on these results, with possible therapeutic benefits to human reproductive failure and endometrial cancer.
Technical Abstract: Transcriptional regulation of the insulin-like growth factor-binding protein-2 (IGFBP-2) gene is not well understood. In this study, we have identified DNA regions that mediate IGFBP-2 gene transcription in two of the three major cell types of the uterine endometrium. Two clusters of transcriptional start sites [nucleotides -109 to -105 and -96 to -87, relative to the translational initiation codon (+1)] were identified for the pig endometrium-expressed IGFBP-2 gene. Upstream regions of this gene were fused to a luciferase reporter gene and used in transient transfection of primary uterine endometrial glandular epithelial (GE) and stromal (ST) fibroblast cells representative of two stages of porcine early pregnancy (days 12 and 18), and of the human uterine (Hec-1-A) glandular epithelial carcinoma cell line. A small (110 bp) upstream region (-874 to -765) stimulated IGFBP-2 promoter activities in all cell types examined and moreover, enhanced heterologous SV40 promoter activity by 3-5-fold in day 18 primary cells. Two noncontiguous copies of the novel sequence motif TCAGGG within the 110 bp fragment were identified to bind a nuclear protein of ~34 kDa molecular weight present in pig uterine endometrium. This pair of sites was stimulatory or repressive depending upon the stage of pregnancy (day 12 and day 18, respectively) at which the GE primary cells were isolated for transfection. A pair of E-box elements (CANNTG) within the 110 bp region also was stimulatory to IGFBP-2 promoter activity; block-mutation of these converted the 110 bp region into a potent transcriptional silencer in glandular epithelium. Results identify novel DNA motifs within a small portion of the 5' flank of the IGFBP-2 gene that regulate promoter function in uterine endometrium of pregnancy and in uterine epithelial carcinoma cells, and which may underlie cell-type expression of the IGFBP-2 gene.